Characterization of plasmacytoid dendritic cells in the CD4C/HIV transgenic mouse model

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

UV-Induced Melanoma Mouse Model Dependent on Endothelin 3 Over-Expression

Melanoma is one of the most aggressive types of cancer. It originates from the transformation of melanocytes present in the epidermal/dermal junction of the human skin. It is commonly accepted that melanomagenesis is influenced by the interaction of environmental factors, genetic factors, as well as tumor-host interactions. DNA photoproducts induced by UV radiation are, in normal cells, repaired by the nucleotide excision repair (NER) pathway. The prominent role of NER in cancer resistance is well exemplified by patients with Xeroderma Pigmentosum (XP). This disease results from mutations in the components of the NER pathway, such as XPA and XPC proteins. In humans, NER pathway disruption leads to the development of skin cancers, including melanoma. Similar to humans afflicted with XP, Xpa and Xpc deficient mice show high sensibility to UV light, leading to skin cancer development, except melanoma. The Endothelin 3 (Edn3) signaling pathway is essential for proliferation, survival and migration of melanocyte precursor cells. Excessive production of Edn3 leads to the accumulation of large numbers of melanocytes in the mouse skin, where they are not normally found. In humans, Edn3 signaling pathway has also been implicated in melanoma progression and its metastatic potential. The goal of this study was the development of the first UV-induced melanoma mouse model dependent on the over-expression of Edn3 in the skin. The UV-induced melanoma mouse model reported here is distinguishable from all previous published models by two features: melanocytes are not transformed a priori and melanomagenesis arises only upon neonatal UV exposure. In this model, melanomagenesis depends on the presence of Edn3 in the skin. Disruption of the NER pathway due to the lack of Xpa or Xpc proteins was not essential for melanomagenesis; however, it enhanced melanoma penetrance and decreased melanoma latency after one single neonatal erythemal UV dose. Exposure to a second dose of UV at six weeks of age did not change time of appearance or penetrance of melanomas in this mouse model. Thus, a combination of neonatal UV exposure with excessive Edn3 in the tumor microenvironment is sufficient for melanomagenesis in mice; furthermore, NER deficiency exacerbates this process.

Cytotoxicity Evaluation of Si-Diatom Frustules in a Mouse Model

Silica additives in bone substitute materials are topical, clinically interesting and have significant support in the Orthopaedic field. Biosilica, e.g isolated from diatoms, has many advantages over its synthetic counterparts, e.g. it is amorphous, thus will be absorbed by the body, however, issues such as purity, presence of endotoxins and cytotoxicity need to be addressed before it can be further exploited. Biosilica isolated from Cyclotella Meneghiniana was then tested in a mouse model, to test the immunological response, organ toxicity (kidney, spleen, liver) and route of metabolism/excretion of silica. Five-week-old Balb-c mice were injected subcutaneously with a single high dose (50mg/ml) of Si-frustules, Si-frustules + organic linker and vehicle only control. Animals were sacrificed at 1d and 28d. The animal studies were conducted under an ethically approved protocol at Queen’s University, Belfast. The animals showed no adverse stress during the experiment and remained healthy until sacrifice. Blood results using ICP-OES analysis suggest the frustules were metabolized between comparator groups at different rates, and clearly showed elevated levels of silicon in groups injected with frustules relative to control. The histology of organs showed no variation in morphology of mice injected frustules relative compared to the control group.
Acknowledgements: The authors would like to thank Marie Curie International Outgoing Fellowships from the EU and Beaufort Marine Biodiscovery Award as part of the Marine Biotechnology Ireland Programme for providing financial support to this project.

A New Mouse Model That Spontaneously Develops Chronic Liver Inflammation and Fibrosis

Here we characterize a new animal model that spontaneously develops chronic inflammation and fibrosis in multiple organs, the non-obese diabetic inflammation and fibrosis (N-IF) mouse. In the liver, the N-IF mouse displays inflammation and fibrosis particularly evident around portal tracts and central veins and accompanied with evidence of abnormal intrahepatic bile ducts. The extensive cellular infiltration consists mainly of macrophages, granulocytes, particularly eosinophils, and mast cells. This inflammatory syndrome is mediated by a transgenic population of natural killer T cells (NKT) induced in an immunodeficient NOD genetic background. The disease is transferrable to immunodeficient recipients, while polyclonal T cells from unaffected syngeneic donors can inhibit the disease phenotype. Because of the fibrotic component, early on-set, spontaneous nature and reproducibility, this novel mouse model provides a unique tool to gain further insight into the underlying mechanisms mediating transformation of chronic inflammation into fibrosis and to evaluate intervention protocols for treating conditions of fibrotic disorders.

Cuminum cyminum Linn (Apiaceae) extract attenuates MPTP-induced oxidative stress and behavioral impairments in mouse model of Parkinson’s disease

Purpose: To evaluate the protective effects of Cuminum cyminum Linn (Apiaceae, CCY) against 1- methyl-4 phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced oxidative stress and behavioral impairments in mouse model of Parkinson’s disease (PD). Methods: MPTP-intoxicated mice model of PD was used for evaluating the effect of CCY extract on behavioral deficits through rota rod, passive avoidance and open field tasks. The effect of CCY extract on oxidative stress levels were assessed by estimating enzyme status, including superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation(LPO) in brain tissues of MPTP-induced mice. Results: MPTP (25 mg/kg, i.p.)-treated mice resulted in a significant (p < 0.001) behavioral deficit in locomotor behavior (from 56.24 ± 1.21 to 27.64 ± 0.94) and cognitive functions (from 298 ± 3.68 s to 207.28 ± 4.12 s) compared with their respective control groups. Administration of CCY extract (100, 200 and 300 mg/kg, p.o.) for three weeks significantly and dose-dependently improved (p < 0.001 at 300 mg/kg) locomotor and cognitive deficits in MPTP-treated mice. CCY treatment also significantly (p < 0.001 at 300 mg/kg) inhibited MPTP-induced decrease in antioxidant enzyme levels (superoxide dismutase and catalase) and lipid peroxides in mice brain tissues. Conclusion: CCY extract exhibits strong protection against MPTP-induced behavioral deficit through enhancement of antioxidant defense mechanisms. Therefore, CCY may be developed as a therapeutic strategy in the treatment of neurodegeneration seen in PD.

Obstructive sleep apneas naturally occur in mice during REM sleep and are more prevalent in a mouse model of Down syndrome

Study Objectives. The use of mouse models in sleep apnea research is limited by the belief that central (CSA) but not obstructive sleep apneas (OSA) occur in rodents. With this study we wanted to develop a protocol to look for the presence of OSAs in wild-type mice and, then, to apply it to a mouse model of Down Syndrome (DS), a human pathology characterized by a high incidence of OSAs. Methods. Nine C57Bl/6J wild-type mice were implanted with electrodes for electroencephalography (EEG), neck electromyography (nEMG), diaphragmatic activity (DIA) and then placed in a whole-body-plethysmographic (WBP) chamber for 8h during the resting (light) phase to simultaneously record sleep and breathing activity. The concomitant analysis of WBP and DIA signals allowed the discrimination between CSA and OSA. The same protocol was then applied to 12 Ts65Dn mice (a validated model of DS) and 14 euploid controls. Results. OSAs represented about half of the apneic events recorded during rapid-eye-movement sleep (REMS) in each experimental group while almost only CSAs were found during non-REMS. Ts65Dn mice had similar rate of apneic events than euploid controls but a significantly higher occurrence of OSAs during REMS. Conclusions. We demonstrated for the first time that mice physiologically exhibit both CSAs and OSAs and that the latter are more prevalent in the Ts65Dn mouse model of DS. These findings indicate that mice can be used as a valid tool to accelerate the comprehension of the pathophysiology of all kind of sleep apnea and for the development of new therapeutical approaches to contrast these respiratory disorders.

Modeling Alzheimer disease with iPSCs and mouse model for the identification of risk factors, new targets, and potential therapeutical strategies

Alzheimer's disease (AD) is the most common neurodegenerative disease in elderly. Donepezil is the first-line drug used for AD. In section one, the experimental activity was oriented to evaluate and characterize molecular and cellular mechanisms that contribute to neurodegeneration induced by the Aβ1-42 oligomers (Aβ1-42O) and potential neuroprotective effects of the hybrids feruloyl-donepezil compound called PQM130. The effects of PQM130 were compared to donepezil in a murine AD model, obtained by intracerebroventricular (i.c.v.) injection of Aβ1-42O. The intraperitoneal administration of PQM130 (0.5-1 mg/kg) after i.c.v. Aβ1-42O injection improved learning and memory, protecting mice against spatial cognition decline. Moreover, it reduced oxidative stress, neuroinflammation and neuronal apoptosis, induced cell survival and protein synthesis in mice hippocampus. PQM130 modulated different pathways than donepezil, and it is more effective in counteracting Aβ1-42O damage. The section two of the experimental activity was focused on studying a loss of function variants of ABCA7. GWA studies identified mutations in the ABCA7 gene as a risk factor for AD. The mechanism through which ABCA7 contributes to AD is not clear. ABCA7 regulates lipid metabolism and critically controls phagocytic function. To investigate ABCA7 functions, CRISPR/Cas9 technology was used to engineer human iPSCs and to carry the genetic variant Y622*, which results in a premature stop codon, causing ABCA7 loss-of-function. From iPSCs, astrocytes were generated. This study revealed the effects of ABCA7 loss in astrocytes. ABCA7 Y622* mutation induced dysfunctional endocytic trafficking, impairing Aβ clearance, lipid dysregulation and cell homeostasis disruption, alterations that could contribute to AD. Though further studies are needed to confirm the PQM130 neuroprotective role and ABCA7 function in AD, the provided results showed a better understanding of AD pathophysiology, a new therapeutic approach to treat AD, and illustrated an innovative methodology for studying the disease.

Longitudinal Micro CT-driven biomarkers as a tool to evaluate lung fibrosis progression and antifibrotic efficacy in a refined Bleomycin mouse model

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with no curative pharmacological treatment. Animal models play an essential role in revealing molecular mechanisms involved in the pathogenesis of the disease. Bleomycin (BLM)-induced lung fibrosis is the most widely used and characterized model for anti-fibrotic drugs screening. However, several issues have been reported, such as the identification of an optimal BLM dose and administration scheme as well as gender-specificity. Moreover, the balance between disease resolution, an appropriate time window for therapeutic intervention and animal welfare remains critical aspects yet to be fully elucidated. In this thesis, Micro CT imaging has been used as a tool to identify the ideal BLM dose regimen to induce sustained lung fibrosis in mice as well as to assess the anti-fibrotic effect of Nintedanib (NINT) treatment upon this BLM administration regimen. In order to select the optimal BLM dose scheme, C57bl/6 male mice were treated with BLM via oropharyngeal aspiration (OA), following either double or triple BLM administration. The triple BLM administration resulted in the most promising scheme, able to balance disease resolution, appropriate time-window for therapeutic intervention and animal welfare. The fibrosis progression was longitudinally assessed by micro-CT every 7 days for 5 weeks after BLM administration and 5 animals were sacrificed at each timepoint for the BALF and histological evaluation. The antifibrotic effect of NINT was assessed following different treatment regimens in this model. Herein, we have developed an optimized mouse model of pulmonary fibrosis, enabling three weeks of the therapeutic window to screen putative anti-fibrotic drugs. micro-CT scanning, allowed us to monitor the progression of lung fibrosis and the therapeutical response longitudinally in the same subject, drastically reducing the number of animals involved in the experiment.

Advanced studies on two animal models, murine and fish. RadKI21A626T mouse model: new insights into molecular mechanisms of enteric neuro-epithelial pathology. European sea bass: study of the effects of essential oils in different diets on the gastric system.

My PhD research period was focused on the anatomical, physiological and functional study of the gastrointestinal system on two different animal models. In two different contexts, the purpose of these two lines of research was contribute to understand how a specific genetic mutation or the adoption of a particular dietary supplement can affect gastrointestinal function. Functional gastrointestinal disorders are chronic conditions characterized by symptoms for which no organic cause can be found. Although symptoms are generally mild, a small subset of cases shows severe manifestations. This subset of patients may also have recurrent intestinal sub-occlusive episodes, but in absence of mechanical causes. This condition is referred to as chronic intestinal pseudo-obstruction, a rare, intractable chronic disease. Some mutations have been associated with CIPO. A novel causative RAD21 missense mutation was identified in a large consanguineous family, segregating a recessive form of CIPO. The present thesis was aimed to elucidate the mechanisms leading to neuropathy underlying CIPO via a recently developed conditional KI mouse carrying the RAD21 mutation. The experimental studies are based on the characterization and functional analysis of the conditional KI Rad21A626T mouse model. On the other hand aquaculture is increasing the global supply of foods. The species selected and feeds used affects the nutrients available from aquaculture, with a need to improve feed efficiency, both for economic and environmental reasons, but this will require novel innovative approaches. Nutritional strategies focused on the use of botanicals have attracted interest in animal production. Previous research indicates the positive results of using essential oils (EOs) as natural feed additives for several farmed animals. Therefore, the present study was designed to compare the effects of feed EO supplementation in two different forms (natural and composed of active ingredients obtained by synthesis) on the gastric mucosa in European sea bass.

Pathogenetic mechanisms of gastrointestinal symptoms in a mouse model of Fabry disease: insights on the microbiota-gut-brain axis

Fabry disease (FD), X-linked metabolic disorder caused by a deficiency in α-galactosidase A activity, leads to the accumulation of glycosphingolipids, mainly Gb3 and lyso-Gb3, in several organs. Gastrointestinal (GI) symptoms are among the earliest and most common, strongly impacting patients’ quality of life. However, the origin of these symptoms and the exact mechanisms of pathogenesis are still poorly understood, thus the pressing need to improve their knowledge. Here we aimed to evaluate whether a FD murine model (α-galactosidase A Knock-Out) captures the functional GI issues experienced by patients. In particular, the potential mechanisms involved in the development and maintenance of GI symptoms were explored by looking at the microbiota-gut-brain axis involvement. Moreover, we sought to examine the effects of lyso-Gb3 on colonic contractility and the intestinal epithelium and the enteric nervous system, which together play important roles in regulating intestinal ion transport and fluid and electrolyte homeostasis. Fabry mice revealed visceral hypersensitivity and a diarrhea-like phenotype accompanied by anxious-like behavior and reduced locomotor activity. They reported also an imbalance of SCFAs and an early compositional and functional dysbiosis of the gut microbiota, which partly persisted with advancing age. Moreover, overexpression of TRPV1 was found in affected mice, and partial alteration of TRPV4 and TRPA1 as well, identifying them as possible therapeutic targets. The Ussing chamber results after treatment with lyso-Gb3 showed an increase in Isc (likely mediated by HCO3- ions movement) which affects neuron-mediated secretion, especially capsaicin- and partly veratridine-mediated. This first characterization of gut-brain axis dysfunction in FD mouse provides functional validation of the model, suggesting new targets and possible therapeutic approaches. Furthermore, lyso-Gb3 is confirmed to be not only a marker for the diagnosis and follow-up of FD but also a possible player in the alteration of the FD colonic ion transport process.

Cardiac functional and structural abnormalities in a mouse model of CDKL5 deficiency disorder

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a rare and severe neurodevelopmental disease that mostly affects girls who are heterozygous for mutations in the X-linked CDKL5 gene. The lack of CDKL5 protein expression or function leads to the appearance of numerous clinical features, including early-onset seizures, marked hypotonia, autistic features, and severe neurodevelopmental impairment. Mouse models of CDD, Cdkl5 KO mice, exhibit several behavioral phenotypes that mimic CDD features, such as impaired learning and memory, social interaction, and motor coordination. CDD symptomatology, along with the high CDKL5 expression levels in the brain, underscores the critical role that CDKL5 plays in proper brain development and function. Nevertheless, the improvement of the clinical overview of CDD in the past few years has defined a more detailed phenotypic spectrum; this includes very common alterations in peripheral organ and tissue function, such as gastrointestinal problems, irregular breathing, hypotonia, and scoliosis, suggesting that CDKL5 deficiency compromises not only CNS function but also that of other organs/tissues. Here we report, for the first time, that a mouse model of CDD, the heterozygous Cdkl5 KO (Cdkl5 +/-) female mouse, exhibits cardiac functional and structural abnormalities. The mice also showed QTc prolongation and increased heart rate. These changes correlate with a marked decrease in parasympathetic activity to the heart and in the expression of the Scn5a and Hcn4 voltage-gated channels. Moreover, the Cdkl5 +/- heart shows typical signs of heart aging, including increased fibrosis, mitochondrial dysfunctions, and increased ROS production. Overall, our study not only contributes to the understanding of the role of CDKL5 in heart structure/function but also documents a novel preclinical phenotype for future therapeutic investigation.

Microenvironment and therapeutic modulation of myelofibrosis in an experimental mouse model.

Primary myelofibrosis(PMF) is the most severe form of Philadelphia-negative myeloproliferative neoplasms(MPNs), characterized by splenomegaly, extramedullary hematopoiesis and bone marrow(BM) fibrosis, with disease progression to leukemia and low survival. The best therapy currently available includes treatment with a JAK inhibitor(Ruxolitinib), which only ameliorates symptoms. Unfortunately, the pathogenesis of the disease is still poorly understood. It has been hypothesized that its progression may be determined by the presence of inflammatory cytokines produced by the bone marrow microenvironment that promote fibrosis. The three aims of this PhD thesis, using the Gata1low mouse model of myelofibrosis, were: 1. Investigate the presence of different cytokines in the bone marrow microenvironment; 2. Test the efficacy of treatment with Reparixin, a CXCR1/2 receptor inhibitor; 3. Test the efficacy of treatment with RB40.34 (P-selectin inhibitor), alone and in combination with Ruxolitinib. In the first study, we demonstrated by immunohistochemistry(IHC) the presence in the BM of Gata1low mice of elevated levels of CXCL1, and its receptors CXCR1/2, and TGF-β1. Particularly, the cells with higher expression of these cytokines were the megakaryocytes. In the second study, we found that treatment with Reparixin in Gata1low mice showed dose-dependent efficacy in reducing bone marrow and splenic fibrosis. Furthermore, by IHC analysis we demonstrated that the treatment induced a decrease in the expression of TGF-β1. In the third study, we found that treatment with RB40.34 in combination with Ruxolitinib normalizes the phenotype of Gata1low mice, reducing fibrosis and the content of TGF-β and CXCL1 in the bone marrow, and restoring the architecture of hematopoiesis in the bone marrow and spleen. In summary, these data provide preclinical evidence that treatment with Reparixin and RB40.34 in combination with Ruxolitinib are effective on reversing the myelofibrotic trait in the Gata1low mouse model and encourage clinical trials to validate these compounds in human patients with PMF.

Pharmacological targeting of P-selectin to halt the progression of myelofibrosis in the Gata1low mouse model

Primary myelofibrosis is a clonal hematopoietic disorder characterized by marked degrees of systemic inflammation. The release of pro-inflammatory factors by clonal hematopoietic cell populations cause the remodeling of a specialized microenvironment, defined niche, in which the hematopoietic stem cells reside. The main source of pro-inflammatory cytokines is represented by malignant megakaryocytes. The bone marrow and spleen from myelofibrosis patients, as well as those from the Gata1low mouse model of the disease, contain increased number of abnormal megakaryocytes. These cells express on their surface high levels of the adhesion receptor P-selectin that, by triggering a pathological megakaryocyte-neutrophil emperipolesis, lead to increased bioavailability of TGF-β1 in the microenvironment and disease progression. Gata1low mice develop with age a phenotype similar to that of patients with myelofibrosis. We previously demonstrated that deletion of the P-selectin gene in Gata1low mice prevented the development of the myelofibrotic phenotype in these mice. In the current study, we tested the hypothesis that pharmacological inhibition of P-selectin may rescue the fibrotic phenotype of Gata1low mice. To test this hypothesis, we have investigated the phenotype expressed by old Gata1low mice treated with the anti-mouse monoclonal antibody against P-selectin RB40.34, alone or in combination with the JAK2 inhibitor Ruxolitinib. The results showed that the combined therapy normalized the phenotype of Gata1low mice with limited toxicity by reducing fibrosis, TGF-β1 and CXCL1 content in the BM and spleen and by restoring hematopoiesis in the bone marrow and the normal architecture of the spleen. In conclusion, pharmacological inhibition of P-selectin was effective in targeting malignant megakaryocytes and the microenvironmental abnormalities that affect the hematopoietic stem cell compartment in this model. These results suggest that P-selectin and JAK1/2 inhibitors in combination may represent a valid therapeutic option for patients with myelofibrosis.

Respiratory allergy to Blomia tropicalis: Immune response in four syngeneic mouse strains and assessment of a low allergen-dose, short-term experimental model

Background: The dust mite Blomia tropicalis is an important source of aeroallergens in tropical areas. Although a mouse model for B. tropicalis extract (BtE)-induced asthma has been described, no study comparing different mouse strains in this asthma model has been reported. The relevance and reproducibility of experimental animal models of allergy depends on the genetic background of the animal, the molecular composition of the allergen and the experimental protocol. Objectives: This work had two objectives. The first was to study the anti-B. tropicalis allergic responses in different mouse strains using a short-term model of respiratory allergy to BtE. This study included the comparison of the allergic responses elicited by BtE with those elicited by ovalbumin in mice of the strain that responded better to BtE sensitization. The second objective was to investigate whether the best responder mouse strain could be used in an experimental model of allergy employing relatively low BtE doses. Methods: Groups of mice of four different syngeneic strains were sensitized subcutaneously with 100 mu g of BtE on days 0 and 7 and challenged four times intranasally, at days 8, 10, 12, and 14, with 10 mu g of BtE. A/J mice, that were the best responders to BtE sensitization, were used to compare the B. tropicalis-specific asthma experimental model with the conventional experimental model of ovalbumin (OVA)-specific asthma. A/J mice were also sensitized with a lower dose of BtE. Results: Mice of all strains had lung inflammatory-cell infiltration and increased levels of anti-BtE IgE antibodies, but these responses were significantly more intense in A/J mice than in CBA/J, BALB/c or C57BL/6J mice. Immunization of A/J mice with BtE induced a more intense airway eosinophil influx, higher levels of total IgE, similar airway hyperreactivity to methacholine but less intense mucous production, and lower levels of specific IgE, IgG1 and IgG2 antibodies than sensitization with OVA. Finally, immunization with a relatively low BtE dose (10 mu g per subcutaneous injection per mouse) was able to sensitize A/J mice, which were the best responders to high-dose BtE immunization, for the development of allergy-associated immune and lung inflammatory responses. Conclusions: The described short-term model of BtE-induced allergic lung disease is reproducible in different syngeneic mouse strains, and mice of the A/J strain was the most responsive to it. In addition, it was shown that OVA and BtE induce quantitatively different immune responses in A/J mice and that the experimental model can be set up with low amounts of BtE.